The present invention relates to monoclonal antibodies and humanized antibodies which bind specifically to a human interleukin-5 receptor xcex1 chain and which are therefore useful for diagnosis or treatment of diseases such as chronic bronchial asthma. The invention also relates to hybridomas and transformants which produce the antibodies, a method for detecting an interleukin-5 receptor xcex1 chain immunologically by means of the monoclonal antibodies and humanized antibodies, as well as a method for diagnosing and treating diseases such as chronic bronchial asthma by means of the monoclonal antibodies and humanized antibodies.
Interleukin-5 (hereinafter referred to a xe2x80x9cIL-5xe2x80x9d) is a kind of lymphokine which is secreted by T cells, mast cells and other cells. Murine IL-5 is known to act as a differentiation and growth factor for B cells and eosinophils. Human IL-5 is known to act mainly as a differentiation and growth factor for eosinophils (Advances in Immunology, 57, 145 (1994); Blood, 79, 3101 (1992)). IL-5 exhibits its action through a specific receptor (IL-5 receptor) which is expressed on the surface of a cell such as eosinophil. It has been shown that human and murine IL-5 receptors (hereinafter referred to as xe2x80x9cIL-5Rsxe2x80x9d) are both composed of two different kinds of proteins, an xcex1 chain (hereinafter referred to as xe2x80x9cIL-5R xcex1xe2x80x9d) and a xcex2 chain (hereinafter referred to as xe2x80x9cIL-5R xcex2xe2x80x9d). In addition, it is known that the binding of IL-5 to IL-5R is via IL-5R xcex1 and that IL-5R xcex2 alone can not bind to IL-5 (EMBO J., 9, 4367 (1990); ibid., 10, 2833 (1991); J. Exp. Med., 177, 1523 (1993); ibid., 175, 341 (1992); Cell, 66, 1175 (1991), Proc. Natl. Acad. Sci., 89, 7041 (1992)). Furthermore, IL-5R xcex2 is known to be a component of receptors for interleukin-3 (hereinafter referred to as xe2x80x9cIL-3xe2x80x9d), granulocyte macrophage colony-stimulating factor and others (hereinafter referred to as xe2x80x9cGM-CSFxe2x80x9d) (Proc. Natl. Acad. Sci., 87.9655 (1990); Cell, 66, 1165 (1991)).
Eosinophils are known to increase in allergic diseases represented by chronic bronchial asthma. Significant infiltration of eosinophils is observed in airways of a patient with chronic bronchial asthma. Eosinophil contains a cytotoxic granular proteins whose deposit is observed in airway tissues of a patient with chronic bronchial asthma or at lesion sites of a patient with atopic dermatitis. These facts suggest that eosinophil plays an important role in the pathogenesis of allergic disorders such as chronic bronchial asthma, atopic dermatitis and the like (Adv. Immunol., 39, 177 (1986); Immunol. Today, 13, 501 (1992)). Hence, studying the kinetics of eosinophils is useful for clinical diagnosis. On the other hand, human IL-5 acts specifically on eosinophils, so IL-5R is believed to be expressed specifically in eosinophils and can therefore be used as a marker specific to human eosinophils. Furthermore, IL-5xcex2 is a receptor for cytokines such as IL-3, GM-CSF and others, so IL-5R xcex1 is believed to be a marker specific to eosinophils. Hence, eosinophils can be detected specifically by immunocyte staining using an anti-human IL-5R xcex1 chain antibody (hereinafter referred to as xe2x80x9canti-hIL-5Rxcex1 antibodyxe2x80x9d). However, no anti-hIL-5R xcex1 antibody is presently known that is capable of specific detection of eosinophils.
Significant eosinophilia was observed in IL-5 transgenic mice (J. Exp. Med., 172, 1425 (1990); ibid. 173, 429 (1991); Int. Immunol., 2, 965 (1990)). Eosinophil infiltration in tissues was suppressed by the administration of an anti-IL-5 antibody in animal models of asthma (Am. Rev. Resir. 147, 548 (1993); ibid., 148, 1623 (1993)). These phenomena indicate that IL-5 actually plays an important role in eosinophilia and the infiltration of eosinophils in vivo. It is also reported that IL-5 is expressed in airway mucosal tissues of a human patient with chronic bronchial asthma and at lesion sites of a patient with atopic dermatitis (J. Clin. Invest., 87, 1541 (1991); J. Exp. Med., 173, 775 (1991)). Further investigations demonstrate that IL-5 exhibits in vitro viability-enhancing action on human eosinophils (J. Immunol., 143, 2311 (1989)) and that IL-5 is an eosinophil-selective activator (J. Exp. Med.,167, 219 (1988)).
Hence, antibodies that bind to IL-5R and which can inhibit the biological activity of IL-5 are expected to inhibit the activity of eosinophil, thus being useful in the treatment of allergic diseases such as chronic bronchial asthma. Anti-mouse IL-5R xcex1 antibodies which can inhibit the biological activity of IL-5 were produced by using as an antigen those IL-5-dependent cells which express a large number of murine IL-5R on their surfaces (Kokai (Japanese published unexamined patent application) No. 108497/91; Int. Immunol., 2, 181 (1990)). However, in the case of humans, no cells are known which express a large number of IL-5R and the expression of IL-5R is reported to be very low in eosinophils (Cell. Immunol., 133, 484 (1991)). Hence, anti-human IL-5R xcex1 antibodies having comparable functions to anti-mouse IL-5R xcex1 antibodies are difficult to produce by methods similar to those for producing the latter. An antibody designated as xe2x80x9cxcex116xe2x80x9d is disclosed as an antibody against human IL-5R xcex1 in EMBO J., 14, 3395 (1995) but this antibody does not have any neutralization activity for IL-5R xcex1.
Human IL-5R xcex1 gene was obtained by preparing a cDNA library from human eosinophil (J. Exp. Med., 175, 341 (1992)) or a human promyelocytic cell HL-60 (Cell, 66, 1175 (1991); Kokai No. 78772/94) and screening the library using as a probe an oligo DNA which had been synthesized on the basis of cDNA of murine IL-5R xcex1 or a partial amino acid sequence of murine IL-5R xcex1 (Kokai No. 54690/94, EMBO J., 9, 4367 (1990)). The transfer of the cDNA into a host cell resulted in the creation of a cell having hIL-5R xcex1 expressed on its surface but the expression level of hIL-5R in this cell was very low (xe2x89xa6104 molecules) (J. Exp. Med., 177, 1523 (1993)). Hence, if one attempts to produce anti-hIL-5R xcex1 antibodies by using this cell as an immunogen, he will find that the relative amount of hIL-5R xcex1 is very small, compared with those of proteins from a host cell and that the absolute protein amount of hIL-5R xcex1 is also very small. In addition, approximately 80% homology at an amino acid level is observed between murine IL-5R xcex1 and human IL-5R xcex1 and murine IL-5 can bind to human IL-5R with high affinity (J. Exp. Med.,175, 341 (1992)). These facts suggest that human IL-5R xcex1 has a lower immunogenicity for mice or rats which are commonly used as animals to be immunized. In fact, almost all of our attempts to prepare anti-hIL-5R xcex1 antibodies using hIL-5R xcex1-expressing cells as an immunogen resulted in a failure.
In the cloning of IL-5R cDNA from a cDNA library of human eosinophil, cDNA encoding soluble human IL-5R xcex1 (hereinafter referred to as xe2x80x9cshIL-5R xcex1xe2x80x9d) has been obtained which corresponds to the N-terminal amino acid sequence (1-313) of IL-5R xcex1 which is defective in the transmembrane region and onwards (J. Exp. Med.,175, 341 (1992)). When shIL-5R xcex1 is used as an immunogen to produce an anti-hIL-5R xcex1 antibody, the shIL-5R xcex1 should have the same three dimensional conformation as that of IL-5R xcex1 expressed on the cell surface and it should be one secreted and produced by a eukaryotic host cell in order to obtain an anti-hIL-5R xcex1 antibody which can inhibit the biological activity of IL-5. In addition, it has been found that the production efficiency of a protein varies significantly depending on the signal peptide (Protein, Nucleic Acid and Enzyme, 35, 2584 (1990)), so it is necessary to select an appropriate signal peptide for secretion and production of the protein.
As mentioned above, it has been found that mRNA which is believed to encode only shIL-5R xcex1 is expressed in eosinophils. It has been confirmed that murine IL-5R is expressed not only in eosinophils but also in B cells and that mRNA which is believed to encode only an extracellular region of IL-5R xcex1 (hereinafter referred to as xe2x80x9csmIL-5R xcex1xe2x80x9d) is expressed in those cells as well as in the case of humans. In addition, it has been reported that smIL-5R xcex1 was detected in blood of mice transplanted with IL-5R expressing murine chronic B cell leukemia cell line (BCL1) or model mice of human autoimmune diseases (J. Immunol. Method, 167, 289 (1994)). These suggest the possibility that the increase in the number of IL-5R expressing cells and their activation may be reflected in the amount of smIL-5R xcex1 secreted in blood. Human IL-5R is believed to be expressed in eosinophils in a limited amount and the increase in the number of eosinophils and their activation may be potentially reflected in the amount of shIL-5R xcex1 in blood. Hence, the quantitative determination of shIL-5R xcex1 is expected to be useful in clinical diagnosis.
Any isolated monoclonal antibody which binds specifically to human IL-5R xcex1 is believed to be useful in the diagnosis and treatment of allergic diseases. However, it should be noted that if a non-human animal-derived monoclonal antibody is administered to a human, it is generally recognized as a foreign matter such that an antibody against the non-human animal-derived monoclonal antibody is produced in the human body, a reaction with the administered non-human animal-derived monoclonal antibody occurs to cause a side effect (J. Clin. Oncol., 2, 881 (1984); Blood, 65, 1349 (1985); J. Natl. Cancer Inst., 80, 932 (1988); Proc. Natl. Acad. Sci., 82, 1242 (1985)), premature clearance of the non-human animal-derived monoclonal antibody occurs (J. Nucl. Med.,26, 1011 (1985); Blood, 65, 1349 (1985); J. Natl. Cancer Inst., 80, 937 (1988)), or therapeutic effect of the monoclonal antibody is reduced (J. Immunol.,135, 1530 (1985); Cancer Res., 46, 6489 (1986)).
In order to solve these problems, attempts have been made to convert non-human animal-derived monoclonal antibodies to human chimeric antibodies or human CDR-grafted antibodies (reconstituted human antibodies) by gene recombinant techniques. A human chimeric antibody is an antibody of which the variable region (hereinafter referred to as xe2x80x9cV regionxe2x80x9d) is derived from a non-human animal antibody and the constant region (hereinafter referred to as xe2x80x9cC regionxe2x80x9d) is derived from a human antibody (Proc. Natl. Acad. Sci., 81, 6851 (1984)). It has been reported that when a human chimeric antibody is administered to a human, antibodies are hardly produced against the non-human animal-derived monoclonal antibody and a half-life in blood is increased by a factor of 6 (Proc. Natl. Acad. Sci.,86, 4220 (1989)). A human CDR-grafted antibody is a human antibody of which the CDR (complementarity determining region) is replaced with the CDR of a non-human animal-derived antibody (Nature, 321, 522 (1986)). It has been reported with experiments on monkeys that a human CDR-grafted antibody has a lower immunogenicity, with the half-life in blood being increased by a factor of 4-5 compared with a mouse antibody (J. Immunol.,147, 1352 (1991)). However, there is no report about a humanized antibody against hIL-5R xcex1.
When a humanized antibody which binds specifically to human IL-5R xcex1 is administered to a human, it is expected to cause no production of an antibody against a non-human animal-derived monoclonal antibody, thereby reducing the side effect and prolonging the half-life in blood, which eventually leads to a high therapeutic effect against allergic diseases such as chronic bronchial asthma, atopic dermatitis and the like.
As a result of the recent progresses in protein and genetic engineering, smaller antibody molecules such as single chain antibodies (Science, 242, 423 (1988)) and disulfide stabilized antibodies (Molecular Immunology, 32, 249 (1995)) are being prepared. Since single chain antibodies and disulfide stabilized antibodies have smaller molecular weights than monoclonal antibodies and humanized antibodies, they are effective in transition into tissues and clearance from blood and their application to the imaging technology and the preparation of complexes with toxins are being underway to provide some promise in therapeutic efficacy (Cancer Research, 55, 318 (1995)). If a single chain antibody or a disulfide stabilized antibody which binds specifically to a human IL-5R xcex1 chain is produced, high diagnostic and therapeutic effects against allergic diseases and the like are anticipated. However, there is no report about a single chain antibody and a disulfide stabilized antibody against a human IL-5R xcex1 chain.
The inventors found that antibodies to a hIL-5R xcex1 chain which recognizes an epitope at 1-313 positions of the N-terminal amino acid sequence of the human IL-5R xcex1 chain which corresponds to an extracellular region defective in the transmembrane region and onwards react specifically with a human interleukin-5 receptor a chain upon immunocyte staining and inhibit the biological activity of interleukin-5. These antibodies can be used to diagnose and treat the aforementioned allergic diseases.
Hence, the present invention provides antibodies which react specifically with a human IL-5R xcex1 chain. The antibodies of the present invention include monoclonal antibodies, humanized antibodies, single chain antibodies, disulfide stabilized antibodies and the like. The antibodies of the present invention may be of any kinds, provided that they react specifically with a hIL-5R xcex1 chain. Those produced by the method explained below are preferred. Briefly, hIL-5R xcex1 protein is prepared as an antigen and applied to immunize animals such as mice, rats, hamsters, rabbits and the like used to prepare hybridomas, thereby inducing to plasma cells having an antigen specificity. The plasma cells are fused with myeloma cells to prepare hybridomas which can produce monoclonal antibodies, and the hybridomas are cultured to obtain the desired anti-IL-5R xcex1 monoclonal antibodies. Any monoclonal antibody can be used so long as it recognizes an epitope at 1-313 positions from the N-terminal amino acid of a human IL-5R xcex1 chain and reacts specifically with the human IL-5R xcex1 chain upon immunocyte staining. Alternatively, any monoclonal antibody can be used so long as it recognizes an epitope at 1-313 positions from the N-terminal amino acid of the human IL-5R xcex1 chain and inhibits the biological activity of human IL-5. The former monoclonal antibodies are exemplified by monoclonal antibody KM1257 produced by hybridoma KM1257 (FERM BP-5133). The latter monoclonal antibodies are exemplified by KM1259 produced by hybridoma KM1259 (FERM BP-5134) and KM1486 produced by hybridoma KM1486 (FERM BP-5651).
The monoclonal antibodies of the present invention react immunologically with a human IL-5R xcex1 chain, a cell having a human IL-5R xcex1 chain expressed on the surface, human eosinophil and the like. The monoclonal antibodies of the present invention react immunologically with a soluble human IL-5R xcex1 chain. Hence, the present invention also provides a method for immunologically detecting and determining a human IL-5R xcex1 chain, a, cell having a human IL-5R xcex1 chain expressed on the surface, human eosinophil and a soluble human IL-5R xcex1 chain. The results of the detection and determination can be used in the diagnosis and treatment of allergic diseases such as chronic bronchial asthma, atopic dermatitis and the like.
The present invention also provides humanized antibodies that have lesser side effects with a prolonged half-life than the monoclonal antibodies and which inhibit the biological activity of IL-5 in a more desired way as therapeutics. The term xe2x80x9chumanized antibodyxe2x80x9d of the present invention is the general term for human chimeric antibodies and human CDR-grafted antibodies.
The term xe2x80x9chuman chimeric antibodyxe2x80x9d means an antibody consisting of a variable region in a heavy chain (hereinafter referred to as xe2x80x9cVHxe2x80x9d) and a variable region in a light chain (hereinafter referred to as xe2x80x9cVLxe2x80x9d) of a non-human animal antibody, as well as a constant region in a heavy chain (hereinafter referred to as xe2x80x9cCHxe2x80x9d) and a constant region in a light chain (hereinafter referred to as xe2x80x9cCLxe2x80x9d) of a human antibody. The term xe2x80x9chuman CDR-grafted antibodyxe2x80x9d means an antibody in which CDR sequences of VH and VL of a human antibody are replaced with CDR sequences of VH and VL of a non-human animal antibody, respectively. An anti-hIL-5R xcex1 chain human chimeric antibody which inhibits the biological activity of IL-5 can be expressed and produced by a process comprising the steps of obtaining cDNAs encoding VH and VL from a hybridoma producing an antibody which can inhibit the biological activity of IL-5, inserting the respective cDNAs into a vector for expression in animal cells which contains a gene encoding human antibody CH and human antibody CL to thereby construct a human chimeric antibody expression vector and transfecting the expression vector into an animal cell. The human chimeric antibody and human CDR-grafted antibody of the present invention may be in any immunoglobulin (Ig) classes and are preferably in a class of IgG. In addition, any C region of IgG subclasses of immunoglobulin such as IgG1, IgG2, IgG3 and IgG4 can be used.
Examples of the human chimeric antibody of the present invention include an antibody of which the VH contains the amino acid sequence of SEQ ID NO: 27, CH is human antibody IgG1, VL contains the amino acid sequence of SEQ ID NO: 29, and CL is human antibody xcexa. A specific example is an antibody designated as xe2x80x9cKM1399xe2x80x9d. A specific example of the human chimeric antibody of which the CH is human antibody IgG4 is an antibody designated as xe2x80x9cKM7399xe2x80x9d. KM1399 can be produced, for example, by transformant KM1399 (FERM BP-5650). KM7399 can be produced, for example, by transformant KM7399 (FERM BP-5649).
The anti-hIL-5R xcex1 chain human CDR-grafted antibody which inhibits the biological activity of IL-5 can be expressed and produced by a process comprising the steps of constructing cDNAs encoding a V region in which CDR sequences of VH and VL of any human antibody are replaced with CDR sequences of VH and VL, respectively, of a non-human animal antibody which can inhibit the biological activity of IL-5, inserting the respective cDNAs into a vector for expression in animal cells which contains a gene encoding human antibody CH and human antibody CL to thereby construct a human CDR-grafted antibody expression vector, and transfecting the expression vector into an animal cell. Examples of the human CDR-grafted antibody of the present invention include an antibody of which the VH contains the amino acid sequence of SEQ ID NO: 83, CH is human antibody IgG1, VL contains the amino acid sequence of SEQ ID NO: 71, and CL is human antibody xcexa. A specific example is an antibody designated as xe2x80x9cKM8399xe2x80x9d. A specific example of the human CDR-grafted antibody of which the CH is human antibody IgG4 is an antibody designated as xe2x80x9cKM9399xe2x80x9d. KM8399 can be produced, for example, by transformant KM8399 (FERM BP-5648). KM9399 can be produced, for example, by transformant KM9399 (FERM BP-5647).
The humanized antibody of the present invention reacts immunologically with a human IL-5R xcex1 chain, a cell having a human IL-5R xcex1 chain expressed on the surface, human eosinophil and the like. Hence, the humanized antibody of the present invention can be used in the diagnosis and treatment of allergic diseases such as chronic bronchial asthma, atopic dermatitis and the like.
In addition, the present invention provides single chain antibodies (single chain Fv; hereinafter referred to as xe2x80x9cscFvxe2x80x9d) and disulfide stabilized antibodies (disulfide stabilized Fv; hereinafter referred to as xe2x80x9cdsFvxe2x80x9d) which exhibit an ability to bind to a human IL-5R xcex1 chain.
The term xe2x80x9csingle chain antibody (scFv)xe2x80x9d means a polypeptide represented by formula VH-L-VL or VL-L-VH in which a single chain of VH and a single chain of VL are linked by an appropriate peptide linker (hereinafter referred to as xe2x80x9cLxe2x80x9d). Any anti-human IL-5R xcex1 chain monoclonal antibodies or human CDR-grafted antibodies can be used as VH and VL in the scFv of the present invention.
The term xe2x80x9cdisulfide stabilized antibody (dsFv)xe2x80x9d means an antibody prepared by binding through a disulfide bond two polypeptides in which each one of the amino acid residues in VH and VL is replaced with cysteine residues. The amino acid residues to be replaced with cysteine residues can be selected on the basis of a presumed steric structure of an antibody in accordance with the method described by Reiter et al. (Protein Engineering,7, 697 (1994)). Either a mouse anti-human IL-5R xcex1 chain monoclonal antibodies or a human CDR-grafted antibodies can be used as VH or VL in the disulfide stabilized antibody of the present invention.
The single chain antibody which has an ability to bind to a human EL-5R xcex1 chain can be expressed and produced by a process comprising the steps of obtaining cDNA encoding VH and VL from a hybridoma which produces an antibody reactive with the human IL-5R xcex1 chain, constructing a single chain antibody expression vector, and transfecting the expression vector into an E. coli, yeast or animal cell. Examples of the monoclonal antibody-derived single chain antibody of the present invention include an antibody of which the VH contains the amino acid sequence of SEQ ID NO: 27 and VL contains the amino acid sequence of SEQ ID NO: 29. Examples of the human CDR-grafted antibody-derived single chain antibody of the present invention include an antibody of which the VH contains the amino acid sequence of SEQ ID NO: 83 and VL contains the amino acid sequence of SEQ ID NO: 71.
The disulfide stabilized antibody which has an ability to bind to a human IL-5R xcex1 chain can be expressed and produced by a process comprising the steps of obtaining cDNA encoding VH and VL from a hybridoma which produces an antibody reactive with the human IL-5R xcex1 chain, inserting the cDNA into an appropriate expression vector, and transfecting the expression vector into an E. coli, yeast or animal cell. Examples of the monoclonal antibody-derived single chain antibody of the present invention include an antibody of which the VH contains the amino acid sequence of SEQ ID NO: 27 and VL contains the amino acid sequence of SEQ ID NO: 29. Examples of the human CDR-grafted antibody-derived disulfide stabilized antibody of the present invention include an antibody of which the VH contains the amino acid sequence of SEQ ID NO: 83 and VL contains the amino acid sequence of SEQ ID NO: 71.
A method for producing an anti-human IL-5R xcex1 chain monoclonal antibody which reacts specifically with a human IL-5R xcex1 chain or which inhibits the biological activity of human IL-5, and a method for producing an anti-human IL-5R xcex1 chain humanized antibody, an anti-human IL-5R xcex1 chain single chain antibody and an anti-human IL-5R xcex1 chain disulfide stabilized antibody all of which inhibit the biological activity of human IL-5, as well as a method for detecting and determining a human interleukin-5 receptor xcex1 chain by means of said antibodies will now be explained in detail.
1. Production of Anti-hIL-5R xcex1 Monoclonal Antibody
(1) Preparation of Antigen
A cell having hIL-5R xcex1 expressed on the cell surface or a cell membrane fraction thereof, or an hIL-5R xcex1-expressing cell CTLL-2 (h5 R) or a cell membrane fraction thereof can be used as an antigen for producing an anti-hIL-5R xcex1 monoclonal antibody. CTLL-2 (h5 R) is an hIL-5R xcex1-expressing cell which was created by inserting a cDNA encoding a full length sequence of a pre-cloned hIL-5Rxcex1 (J. Exp. Med., 175, 341 (1992)) into an expression vector for animal cells such as pCAGGS (Gene,108, 193 (1991)) and transfecting the expression vector into murine T cell line CTLL-2.
For expression in a prokaryotic host cell such as E. coli, a full length or partial fragment of cDNA encoding hIL-5R xcex1 can be inserted into an expression vector such as commercially available pGEX (Pharmacia), pET system (Novagen), pMKex1 to be described in section (11) of Example 1 below or the like and the full length hIL-5R xcex1 sequence or a partial fragment thereof can be expressed either as such or as a fusion protein. After disruption of the cell, the protein expressed by E. coli can be purified by SDS-polyacrylamide electrophoresis, affinity chromatography based on the nature of the fusion protein, or the like.
In the method of expressing the full length IL-5R xcex1 sequence or a partial fragment thereof either as such or as a fusion protein, eukaryotic host cells such as insect cells, mammalian cells and the like can be used.
In the case of using a mammalian cell, a full length or a partial fragment of cDNA encoding hIL-5R xcex1 is inserted into a vector such as pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (J. Biochem.,101, 1307 (1987)), pAGE210 to be described in section (1) of Example 1 below or the like to thereby construct an expression vector for the protein. In order to express efficiently the full length hIL-5R xcex1 sequence encoded by the cDNA or a partial fragment thereof either as such or as a fused protein, the nucleotide sequence encoding a signal peptide in the cDNA is preferably replaced by the nucleotide sequence encoding a signal peptide of a protein which can be expressed at a high level in a eukaryotic host cell. Known signal peptides of proteins including those of human growth hormone, anti-ganglioside GD3 chimeric antibody KM871 (Kokai No. 304989/93) and the like are preferably used.
The thus constructed expression vector can be transfected into host cells by a known method such as electroporation (Kokai No. 257891/90; Cytotechnology, 3, 133 (1990)), lipofectin method (Proc. Natl. Acad. Sci., 84, 7413 (1987)) or the like. The cultivation of the cells in an appropriate medium can result in the production of the full length hIL-5R xcex1 sequence or a partial fragment thereof either as such or as a fusion protein in the cells or the culture supernatant. A serum-free medium is preferably used because it can facilitate the purification of the partial fragment or fusion protein of hIL-5R xcex1 produced in the culture supernatant.
In the case of using an insect cell, a full length or a partial fragment of cDNA encoding hIL-5R xcex1 is inserted using a Baculo Gold Starter Kit (Pharmingen) to prepare a recombinant baculovirus and insect cells of Sf9, Sf21 (Pharmingen) or the like are infected with the recombinant virus such that the full length hIL-5R xcex1 sequence or a partial fragment thereof is produced either as such or as a fusion protein in the cells or the culture supernatant (Bio/Technology, 6, 47 (1988)).
The full length hIL-5R xcex1 sequence or a partial fragment or fusion protein thereof produced by the animal or insect cells can be purified from the culture supernatant or the like by a known method of protein purification such as salting-out, affinity chromatography, ion-exchange chromatography or the like and can be used as an antigen. Particularly in the case where the hIL-5R xcex1 is produced as a fusion protein with a constant region of immunoglobulin, it is preferably purified using an affinity column having fixed thereto protein A which has specific affinity for the constant region of immunoglobulin.
(2) Immunization of animal and preparation of antibody-producing cells
Any animal such as mice, rats, hamsters, rabbits and the like can be used as animals to be immunized, provided that they can be used to prepare hybridomas. An embodiment in which mice or rats are used will be explained herein. Mice and rats of 3-20 weeks old are immunized with shIL-5R xcex1 or CTLL-2 which have hIL-5R xcex1 expressed on the surface (J. Exp. Med., 177, 1523 (1993)) as an antigen and antibody-producing cells are collected from the spleens, lymph nodes and peripheral blood of the animals. Immunization is performed by administering the animals with the antigen together with an appropriate adjuvant such as complete Freund""s adjuvant or a combination of aluminum hydroxide gel and pertussis vaccine either subcutaneously, intravenously or intraperitoneally. The antigen is administered 5-10 times at intervals of 1-2 weeks after the first administration. Blood is collected from the ophthal venous plexus at day 3-7 after each administration and the serum is examined for a reactivity with the antigen by enzyme immunoassay (xe2x80x9cEnzyme Immunoassay (ELISA)xe2x80x9d, published by Igakushoin, 1976).
A mouse or rat whose serum shows a satisfactory antibody titer to shIL-5R xcex1 or the cells which have hIL-5R xcex1 expressed on the surface, which are used for immunization, can be used as a source of antibody-producing cells.
In order to perform fusion of a spleen cell with a myeloma cell, the spleen is removed from the immunized mouse at day 3-7 after the final administration of the antigenic substance and spleen cells are collected. The spleen is sliced in an MEM medium (Nissui Pharmaceuticals) and dispersed with a pair of tweezers. After centrifugation (1,200 rpm, 5 min), the supernatant is removed. The precipitate is treated with a Tris-ammonium chloride buffer (pH 7.65) for 1-2 minutes to remove erythrocytes and washed with MEM medium 3 times to prepare splenocytes for use in cell fusion.
(3) Preparation of Myeloma Cells
An established cell line from a mouse or a rat is used as a myeloma cell. Examples include myeloma cell lines P3-X63Ag8-U1 (P3-U1) (Curr. Topics Microbiol. Immunol., 81, 1 (1978); Europ. J. Immunol., 6, 511 (1976)), SP2/0-Ag14 (SP-2) (Nature, 276, 269 (1978)), P3-X63-Ag8653 (653) (J. Immunol., 123, 1548 (1979)) and P3-X63-Ag8 (X63) (Nature,256, 495 (1975)) which are derived from 8-azaguanine-tolerant mice (BALB/c). These cell lines can be subcultured in 8-azaguanine medium which is RPMI-1640 medium supplemented with glutamine (1.5 mM), 2-mercaptoethanol (5xc3x97105 M), gentamicin (10 xcexcg/ml) and fetal calf serum (FCS) (CSL, 10%) (hereinafter referred to as xe2x80x9cnormal mediumxe2x80x9d), which is further supplemented with 8-azaguanine (15 xcexcg/ml). They should be subcultured in a normal medium 3-4 days before cell fusion to ensure a cell count of at least 2xc3x97107 cells on the day of cell fusion.
(4) Cell Fusion
The antibody-producing cells described in 1 (2) and the myeloma cells described in 1 (3) are washed thoroughly with MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of sodium chloride, 1 L of distilled water, pH 7.2). These cells are mixed such that a cell count ratio of the antibody-producing cells to the myeloma cells is 5-10:1. After centrifugation (1,200 rpm, 5 min), the supernatant is removed. The precipitated cells are dispersed and a mixed solution composed of ethylene glycol-1000 (PEG-1000)(2 g), MEM (2 ml) and dimethyl sulfoxide (DMSO) (0.7 ml) is then added to the cells in an amount of 0.2-1 ml/108 antibody-producing cells while stirring. An MEM medium (1-2 ml) is added several times at intervals of 1-2 minutes and an additional MEM medium is then added such that the total volume is 50 ml. After centrifugation (900 rpm, 5 min), the supernatant is removed. The cells are dispersed gently and then suspended gently in 100 ml of a HAT medium (a normal medium supplemented with 10xe2x88x924 M hypoxanthine, 1.5xc3x9710xe2x88x925 M thymidine and 4xc3x9710xe2x88x927 M aminopterin) by suction and blowoff with a pipette.
The cell suspension is dispensed in a 96-well culture plate in an amount of 100 xcexcl/well and cultured in a 5% CO2 incubator at 37xc2x0 C. for 7-14 days.
After the cultivation, an aliquot of the culture supernatant is examined by enzyme immunoassay to be described in 1 (5) to select a well that is reactive specifically with a recombinant protein such as a fusion protein with shIL-5R xcex1 or hIL-5R xcex1 described in 1 (1). Subsequently, cloning by limiting dilution is repeated twice. An aminopterin-free HAT medium is used in the first cloning and a normal medium in the second cloning. A cell exhibiting a high antibody titer stably is selected as a hybridoma cell line which produces a mouse or rat anti-hIL-5R xcex1 monoclonal antibody.
(5) Selection of Mouse or Rat Anti-human IL-5R xcex1 Monoclonal Antibody
A mouse or rat anti-hIL-5R xcex1 monoclonal antibody-producing hybridoma is selected in accordance with a method as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988) by the measurement method described below. By the method, the activity of an anti-hIL-5R xcex1 antibody in the culture supernatant of the transformants producing an anti-hIL-5R xcex1 humanized antibody, a single chain antibody or a disulfide stabilized antibody which are to be described below or the activities of all purified anti-hIL-5R xcex1 antibodies can be determined.
An appropriate plate is coated with shIL-5R xcex1 or a recombinant protein such as a fusion protein with hIL-5R xcex1 described in 1 (1). The plate is reacted with a primary antibody which is the hybridoma culture supernatant or the purified antibody to be obtained in 1 (6) and reacted with a secondary antibody which is an anti-mouse immunoglobulin antibody or an anti-rat immunoglobulin antibody which is labeled with biotin, an enzyme, a chemiluminescent substance or a radioactive compound. Subsequently, a reaction is performed in accordance with the specific kind of the label, whereby a hybridoma that is reactive specifically with hIL-5R xcex1 is selected as a hybridoma producing a mouse anti-hIL-5R xcex1 monoclonal antibody.
If the culture supernatant of the transformants producing an anti-hIL-5R xcex1 humanized antibody, a single chain antibody or a disulfide stabilized antibody, or an antibody purified therefrom is reacted as a primary antibody, an anti-human immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance or a radioactive compound is used as a secondary antibody and a reaction is performed in accordance with the specific kind of the label for detection.
An appropriate plate is coated with shIL-5R xcex1 or a recombinant protein such as a fusion protein with a recombinant protein hIL-5R xcex1 described in 1 (1). Any one of the hybridoma culture supernatant, the culture supernatant of the transformants producing an anti-hIL-5R xcex1 humanized antibody, a single chain antibody or a disulfide stabilized antibody, or an antibody purified therefrom is mixed and reacted with human IL-5 labeled with biotin, an enzyme, a chemiluminescent substance or a radioactive compound. Subsequently, a reaction is performed in accordance with the specific kind of the label so as to determine an activity in inhibiting the binding of human IL-5 to human IL-5R xcex1. This method is used to screen hybridomas for selection of one having a high inhibitory activity against human IL-5.
(6) Production of Mouse or Rat Monoclonal Antibody
A 8-10 week-old mouse or nude mouse is treated with pristane. More specifically, the mouse is administered intraperitoneally with pristane (2,6,10,14-tetramethylpentadecane, 0.5 ml) and bred for 2 weeks. The mouse is administered intraperitoneally with the mouse or rat anti-hIL-5R xcex1 monoclonal antibody-producing hybridoma cell lines (as obtained in 1 (3)) in an amount of 2xc3x97107xe2x88x925xc3x97106 cells/mouse. The hybridoma caused ascites tumor after 10-21 days administration. The ascites is collected from the mouse and centrifuged (3,000 rpm, 5 min) to remove a solid portion. The precipitate is salted out and applied to a column for a caprylic acid precipitation, or a DEAE-Sepharose column, a protein A-column or a Cellulofine GSL2000 column (Biochemical Industry) to collect IgG or IgM fractions. These fractions are used as a purified monoclonal antibody.
The subclass of the antibody is determined using a mouse or rat monoclonal antibody typing kit. The mass of the protein is calculated by a Lowry method or from the absorbance at 280 nm.
2. Production of Anti-human IL-5R xcex1 Humanized Antibody
(1) Construction of Humanized Antibody Expression Vector
In order to produce a humanized antibody from a non-human animal antibody, a humanized antibody expression vector is prepared. The humanized antibody expression vector is a vector for expression in animal cells into which a gene encoding CH and CL, C regions of a human antibody, have been transfected. Such an expression vector is constructed by inserting two genes, one encoding CH of a human antibody and the other encoding CL of a human antibody, into an expression vector for animal cells. Any C regions of a human antibody such as Cxcex31 and Cxcex34 of a human antibody H chain, Cxcexa of a human antibody L chain and the like can be used. A chromosomal DNA consisting of an exon(s) and an intron(s) or cDNA can be used as a gene encoding a C region of a human antibody. Any expression vectors can be used as expression vectors for animal cells, provided that they can incorporate and express a gene encoding a C region of a human antibody. Examples are pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27, 223 (1984)), pKCR (Proc. Natl. Acad. Sci., 78, 1527 (1981)) and pSG1 xcex2d2-4 (Cytotechnology, 4. 173 (1990)). A promoter and an enhancer to be used in preparation of an expression vector for animal cells are exemplified by an SV40 early promoter and enhancer (J. Biochem.,101, 1307 (1987)), a Moloney mouse leukemia virus LTR promoter and enhancer (Biochem. Biophys. Res. Commun., 149, 960 (1987)), an immunoglobulin H chain promoter (Cell, 41, 479 (1985)) and enhancer (Cell, 33, 717 (1983)), and the like.
The humanized antibody expression vector may be either of a type in which a gene encoding an antibody H chain and a gene encoding an antibody L chain exist on separate vectors or of a type in which both genes exist on the same vector (tandem type). In terms of ease of construction of a humanized antibody expression vector, easiness of introduction into animal cells, balance between the expression amounts of antibody H and L chains in the animal cells and for other reasons, a tandem type of humanized antibody expression vector is more preferred (J. Immunol. Methods, 167, 271 (1994)).
(2) Preparation of cDNA Encoding VH and VL of Non-human Animal Antibody
cDNA encoding VH and VL of a non-human animal antibody such as a mouse anti-human IL-5R xcex1 chain monoclonal antibody is obtained, for example, as follows:
mRNA is extracted from an anti-human IL-5R xcex1 chain monoclonal antibody-producing cell such as a mouse anti-human EL-5R xcex1 chain antibody-producing hybridoma and used to synthesize cDNA. The synthesized cDNA is inserted into a vector such as a phage or a plasmid to prepare a cDNA library. From the library, with a portion in a V or C region of a non-human animal antibody such as a mouse antibody being used as a probe, a recombinant phage or plasmid which contains cDNA encoding VH and a recombinant phage or plasmid which contains cDNA encoding VL are isolated separately. The full nucleotide sequences of VH and VL of an antibody of interest which exist on the recombinant phage or plasmid are determined and the full amino acid sequences of the VH and VL are deduced from the nucleotide sequences.
(3) Construction of Human Chimeric Antibody Expression Vector
A human chimeric antibody expression vector can be constructed by inserting cDNA encoding VH and VL of a non-human animal antibody in a region upstream of the gene encoding CH and CL of the human antibody on the humanized antibody expression vector which has been constructed in 2 (1). For example, a restriction enzyme recognition site for cloning of cDNA encoding VH and VL of a non-human animal antibody is created preliminarily in a region upstream of a gene encoding CH and CL of the human antibody on a chimeric antibody expression vector. At the cloning site, cDNA encoding a V region of a non-human animal antibody is inserted through a synthetic DNA (see below) to prepare a human chimeric antibody expression vector. The synthetic DNA consists of a nucleotide sequence at the 3xe2x80x2 end of a V region of the non-human animal and a nucleotide sequence at the 5xe2x80x2 end of a C region of the human antibody and are prepared by a DNA synthesizer such that it has appropriate restriction enzyme sites at both ends.
(4) Identification of CDR Sequences of Non-human Animal Antibody
VH and VL which form an antigen-binding site of an antibody consist of 3 complementarity determining regions (CDRs) having a wide variety of sequences which link the VH and VL to 4 framework regions (hereinafter referred to as R regionsxe2x80x9d) having relatively conserved sequences (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991). The amino acid sequence of the respective CDR (CDR sequence) can be identified by comparison with the amino acid sequences of V regions of known antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991).
(5) Construction of cDNA Encoding V Region of Human CDR-grafted Antibody
cDNA encoding VH and VL of a human CDR-grafted antibody can be obtained as follows:
In the first step, for each of VH and VL, the amino acid sequence of FR in a V region of a human antibody to which CDR in a V region of a non-human animal antibody of interest is to be grafted is selected. Any amino acid sequences of FRs in V regions derived from human antibodies can be used as the amino acid sequences of FRs in V regions of human antibodies. For example, the amino acid sequences of FRs in V regions of human antibodies recorded in Protein Data Bank and amino acid sequences common to subgroups of FRs in V regions of human antibodies (Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991) can be used. In order to produce a human CDR-grafted antibody having an excellent activity, an amino acid sequence having high homology with the amino acid sequence of a V region of a non-human animal antibody of interest is desired. In the second step, a DNA sequence encoding the selected amino acid sequence of FR in a V region of a human antibody is ligated to a DNA sequence encoding the amino acid sequence of CDR in a V region of a non-human animal antibody and a DNA sequence encoding the amino acid sequences of VH and VL is designed. In order to obtain a DNA sequence designed to construct a CDR-grafted antibody variable region gene, several synthetic DNAs are designed for each strand such that the full DNA sequence is covered. Using the synthetic DNAs, polymerase chain reaction (hereinafter referred to as CRxe2x80x9d) is performed. For each strand, preferably 6 synthetic DNAs are designed in view of the reaction efficiency of PCR and the lengths of DNAs which can be synthesized. After the reaction, amplified fragments are subdloned into appropriate vectors and their nucleotide sequences are determined, thereby obtaining a plasmid which contains cDNA encoding the amino acid sequence of a V region of each strand of a human CDR-grafted antibody of interest. Alternatively, cDNA encoding the amino acid sequence of a V region of each strand of a human CDR-grafted antibody of interest may be constructed by synthesizing the full sequences of sense and antisense strands using synthetic DNAs consisting of about 100 bases and subjecting them to annealing and ligation.
(6) Modification of the Amino Acid Sequence of V Region of Human CDR-grafted Antibody
It is known that if a human CDR-grafted antibody is prepared by simply grafting only CDR in a V region of a non-human animal antibody of interest between FRs in a V region of a human antibody, its activity is lower than that of the original non-human animal antibody (BIO/TECHNOLOGY, 9,266 (1991)). Hence, among the amino acid sequences of FR in a V region of a human antibody, an amino acid residue which takes part in direct binding to an antigen, an amino acid residue which interacts with an amino acid residue in CDR, or an amino acid residue which may take part in the maintenance of the steric structure of an antibody is modified to an amino acid residue that is found in the original non-human animal antibody such that the activity of the human CDR-grafted antibody is increased. For efficient identification of the amino acid residue, the steric structure of an antibody is constructed and analyzed by X-ray crystallography, computer-modeling or the like. However, no method for producing a human CDR-grafted antibody which can be applied to any antibodies has yet been established and, therefore, various attempts must currently be made on a case-by-case basis.
The modification of the selected amino acid sequence of FR in a V region of a human antibody can be accomplished using various primers for mutation by PCR described in 2 (5). Amplified fragments obtained by the PCR are subcloned into appropriate vectors and their nucleotide sequences are determined, thereby obtaining a vector containing cDNA into which a mutation of interest has been introduced (hereinafter referred to as xe2x80x9camino acid sequence-replaced vectorxe2x80x9d).
Alternatively, the modification of an amino acid sequence in a narrow region may be accomplished by a PCR-mutagenesis method using primers for mutation consisting of 20-35 bases. More specifically, a sense mutation primer and an antisense mutation primer which consist of 20-35 bases and which contain DNA sequences encoding the amino acid residue to be modified are synthesized and used to perform 2-step PCR using as a template a plasmid which contains cDNA encoding the amino acid sequence of a V region which is to be modified. The finally amplified fragments are subcloned into appropriate vectors and their nucleotide sequences are determined, thereby obtaining an amino acid sequence-modified vector containing cDNA into which a mutation of interest has been introduced.
(7) Construction of Human CDR-grafted Antibody Expression Vector
A human CDR-grafted antibody expression vector can be constructed by inserting the cDNA encoding VH and VL of the human CDR-grafted antibody obtained in 2 (5) and 2 (6) in a region upstream of the gene encoding CH and CL of the human antibody in the humanized antibody expression vector described in 2 (1). For example, if recognition sites for appropriate enzymes are introduced at the ends of the 5xe2x80x2 and 3xe2x80x2 terminal synthetic DNAs during PCR for construction of cDNA encoding the amino acid sequences of VH and VL of the human CDR-grafted antibody, the cDNA can be inserted in a region upstream of a gene encoding a C region of a desired human antibody such that it is expressed in an appropriate form.
(8) Transient Expression of Humanized Antibodies and Evaluation of Their Activities
In order to evaluate the activities of a wide variety of humanized antibodies efficiently, the human chimeric antibody expression vector described in 2 (3), and the human CDR-grafted antibody expression vector described in 2 (7) or their modified vectors may be transfected into COS-7 cells (ATCC CRL1651) and humanized antibodies expressed transiently (Methods in Nucleic Acids Res., CRC Press, p.283, 1991), followed by determination of their activities.
Examples of the method for transfecting the expression vector into a COS-7 cell include a DEAE-dextran method (Methods in Nucleic Acids Res., CRC Press, p.283, 1991), a lipofection method (Proc. Natl. Acad. Sci., 84, 7413 (1987)) and the like.
After transfection of the vector, the activities of the humanized antibodies in the culture supernatant can be determined by the enzyme immunoassay (ELISA) described in 1 (5) and the like.
(9) Stable Expression of Humanized Antibodies and Evaluation of Their Activities
Transformants which produce a humanized antibody stably can be obtained by transfecting into appropriate host cells the human chimeric antibody expression vector described in 2 (3) and the human CDR-grafted antibody expression vector described in 2 (7).
Examples of the method for transfecting the expression vector into host cells include electroporation (Kokai No. 257891/90, Cytotechnology, 3, 133 (1990)) and the like.
Any cells can be used as host cells into which the humanized antibody expression vector is to be transfected, provided that they can express a humanized antibody. Examples are mouse SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cells which are detective in dihydrofolate reductase gene (hereinafter referred to as xe2x80x9cDHFR genexe2x80x9d) (Proc. Natl. Acad. Sci.,77, 4216 (1980)) and rat YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as xe2x80x9cYB2/0 cellxe2x80x9d).
After transfection of the vector, transformants which express a humanized antibody stably are selected in accordance with the method disclosed in Kokai No. 257891/90, using an RPMI1640 medium containing G418 and FCS. The humanized antibody can be produced and accumulated in a culture medium by culturing the selected transformants in a medium. The activity of the humanized antibody in the culture medium is determined by the method described in 1 (5) or the like. The production of the humanized antibody by the transformants can be increased by the method described in Kokai No. 257891/90, utilizing a DHFR gene-amplification system or the like.
The humanized antibody can be purified from the culture supernatant of the transformants by using a protein A column (Antibodies, A Laboratory Manual, Cold Spring Harbor, Chapter 8, 1988). Any other conventional methods for protein purification can be used. For example, the humanized antibody can be purified by a combination of gel filtration, ion-exchange chromatography, ultrafiltration and the like. The molecular weight of the H chain or L chain of the purified humanized antibody or the antibody molecule as a whole is determined by polyacrylamide gel electrophoresis (SDS-PAGE) (Nature, 227, 680, (1970)), western blotting (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 12, 1988) and the like.
The reactivity of the purified humanized antibody and the inhibition activity of the humanized antibody against IL-5 can be determined by the method described in 1 (5).
(10) Method of Use of Humanized Antibody
The humanized antibody of the present invention can bind specifically to a human IL-5R xcex1 chain, thereby inhibiting the biological activity of IL-5. Hence, the humanized antibody of the present invention is expected to inhibit the function of eosinophils which are controlled in differentiation and growth by IL-5. Accordingly, the humanized antibody of the present invention will be useful in the treatment of diseases where eosinophils are associated with their pathogenesis. Since almost all portions of the humanized antibody of the present invention are derived from the amino acid sequence of a human antibody, it is expected not only to exhibit immunogenicity in the human body but also to maintain its effect for a long period of time. The humanized antibody of the present invention can be used either alone or in combination with at least one pharmaceutically acceptable adjuvant. For example, the humanized antibody is dissolved in physiological saline or an aqueous solution of glucose, lactose, mannitol or the like to prepare a pharmaceutical composition. Alternatively, the humanized antibody is lyophilized by a conventional method and sodium chloride is added to prepare an injection in a powder form. If necessary, the present pharmaceutical composition may contain any additive that is well known in the field of pharmaceutical preparations such as a pharmaceutically acceptable salt and the like.
The present pharmaceutical composition can be administered to mammals including human at a dose of 0.1-20 mg/kg/day of the humanized antibody, which may vary depending on the age and conditions of the patient and the like. The administration is given once a day (single dose or continuous administration), 1-3 times a week or once every 2-3 weeks by intravenous injection.
3. Production of Anti-human IL-5R xcex1 Single Chain Antibody
(1) Construction of Single Chain Antibody Expression Vector
A vector for expression of a single chain antibody of a non-human animal antibody or a single chain antibody of a human CDR-grafted antibody can be constructed by inserting into a single chain antibody expression vector the cDNAs encoding VH and VL of a non-human animal antibody or a human CDR-grafted antibody which are described in 2 (2), 2 (5) and 2 (6). Any expression vectors can be used as single chain antibody expression vectors, provided that they can incorporate and express the cDNAs encoding VH and VL of a non-human animal antibody or a human CDR-grafted antibody. Examples are pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27, 223 (1984)), pKCR (Proc. Natl. Acad. Sci., 78, 1527 (1981)) and pSG1 xcex2d2-4 (Cytotechnology, 4. 173 (1990)). A host for use in expressing a single chain antibody can be selected from among E. coli, yeast and animal cells and the like. In this case, an expression vector which is compatible with the specific host should be selected. The single chain antibody can be secreted out of the cell and transported into the periplasm region or retained within the cell by inserting a cDNA encoding an appropriate signal peptide into the expression vector.
A single chain antibody expression vector into which the cDNA encoding a single chain antibody of interest has been inserted can be constructed by inserting the cDNA encoding a single chain antibody consisting of VH-L-VL or VL-L-VH (where L is a peptide linker) into the selected expression vector in a region downstream of an appropriate promoter and a signal peptide.
The cDNA encoding a single chain antibody can be obtained by linking a VH encoding cDNA to a VL encoding cDNA through a synthetic DNA encoding a peptide linker having recognition sites for appropriate restriction enzymes at both the ends. It is important to optimize the linker peptide such that its addition does not interfere with the binding of VH and VL to an antigen. For example, the linker described by Pantoliano et al. (Biochemistry, 30, 10117 (1991)) and its modified versions may be used.
(2) Expression of Single Chain Antibody and Evaluation of its Activity
A transformant which produces a single chain antibody of interest can be obtained by transfecting the single chain antibody expression vector constructed in 3 (1) into an appropriate host cell by electroporation (Kokai No. 257891/90; Cytotechnology, 3, 133 (1990)) or the like. After transfection of the expression vector, the activity of the single chain antibody in the culture supernatant can be determined by the method described in 1 (5) or the like.
The collection and purification of the single chain antibody of the present invention can be accomplished by a combination of known techniques. For example, if the single chain antibody is secreted in a medium, it can be concentrated by ultrafiltration and its collection and purification can be then performed by antigen affinity chromatography or ion-exchange chromatography or gel filtration. If the single chain antibody is transported into the periplasm region of the host cell, it can be concentrated by ultrafiltration following the application of an osmotic shock and its collection and purification can be then performed by antigen affinity chromatography or ion-exchange chromatography or gel filtration. If the single chain antibody is insoluble and exists as a granule (i.e., inclusion body), its collection and purification can be performed by lysis of the cell, repeated centrifugation and washing for isolation of the granule, solubilization with guanidine-HCl, an operation for returning the structure of the single chain antibody to an active structure and the subsequent purification of an active molecule.
The activity of the purified single chain antibody can be determined by the method described in 1 (5) or the like.
(3) Method of Using Single Chain Antibody
The single chain antibody of the present invention can bind specifically to a human IL-5R xcex1 chain, and inhibit the biological activity of IL-5. Hence, the single chain antibody of the present invention is expected to inhibit the function of eosinophils which are controlled in differentiation and growth by IL-5. Accordingly, the single chain antibody of the present invention will be useful in the treatment of diseases in which eosinophils are associated with the pathogenesis. The single chain antibody of the present invention can be used either alone or in combination with at least one pharmaceutically acceptable adjuvant. For example, the single chain antibody is dissolved in physiological saline or an aqueous solution of glucose, lactose, mannitol or the like to prepare a pharmaceutical composition. Alternatively, the single chain antibody is lyophilized by a conventional method and sodium chloride is added to prepare an injection in a powder form. If necessary, the present pharmaceutical composition may contain any additive that is well known in the field of pharmaceutical preparations such as a pharmaceutically acceptable salt and the like.
The present pharmaceutical composition can be administered to mammals, including humans, at a dose of 0.1-20 mg/kg/day of the signal chain antibody, which may vary depending on the age and condition of the patient and the like. The administration is given once a day (single dose or continuous administration), 1-3 times a week or once every 2-3 weeks by intravenous injection.
4. Production of Anti-human IL-5R xcex1 Disulfide Stabilized Antibody
(1) Production of Disulfide Stabilized Antibody
A disulfide stabilized antibody can be produced by a process comprising the steps of providing cDNAs encoding VH and VL of a non-human animal antibody or cDNAs encoding VH and VL of a human CDR-grafted antibody, modifying the DNA sequence which corresponds to a one-amino acid residue at an appropriate position in the respective cDNA with a DNA sequence corresponding to a cysteine residue, expressing the modified cDNAs and purifying the resultant peptide and then forming a disulfide bond. The modification of an amino acid residue to a cysteine residue can be performed by a mutagenesis method using PCR described in 2 (5).
A disulfide stabilized antibody H chain expression vector and a disulfide stabilized antibody L chain expression vector can be constructed by inserting the resulting cDNAs encoding the modified VH and modified VL into appropriate expression vectors. Any expression vectors can be used as disulfide stabilized antibody expression vectors, provided that they can incorporate and express cDNAs encoding a modified VH and a modified VL. For example, pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27, 223 (1984)), pKCR (Proc. Natl. Acad. Sci., 78, 1527 (1981)), pSG1 xcex2d2-4 (Cytotechnology, 4. 173 (1990)) and the like can be used. A host used to express a disulfide stabilized antibody L chain expression vector and a disulfide stabilized antibody H chain expression vector for formation of a disulfide stabilized antibody can be selected from among E. coli, yeast and animal cells, and the like. In this case, an expression vector which is compatible with the specific host should be selected. The disulfide stabilized antibody can be secreted out of the cell and transported into the periplasm region or retained within the cell by inserting a cDNA encoding an appropriate signal peptide into the expression vector.
(2) Expression of Disulfide Stabilized Antibody and Evaluation of its Activity
A transformant which produces a disulfide stabilized antibody H chain or a disulfide stabilized antibody L chain of interest can be obtained by transfecting into a host cell the disulfide stabilized antibody H chain expression vector or the disulfide stabilized antibody L chain expression vector that were constructed in 4 (1) by electroporation (Kokai No. 257891/90; Cytotechnology, 3, 133 (1990)) or the like. After introduction of the expression vector, the expression of the disulfide stabilized antibody H chain or disulfide stabilized antibody L chain in the culture supernatant or the like can be confirmed by the method described in 1 (5).
The collection and purification of the disulfide stabilized antibody H chain or disulfide stabilized antibody L chain can be accomplished by combinations of known techniques. For example, if the disulfide stabilized antibody H chain or disulfide stabilized antibody L chain is secreted in a medium, they can be concentrated by ultrafiltration and their collection and purification can be then performed by various types of chromatography or gel filtration. If the disulfide stabilized antibody H chain or disulfide stabilized antibody L chain is transported into the periplasm region of the host cell, they can be concentrated by ultrafiltration after the application of an osmotic shock to the cell and their collection and purification can be then performed by various types of chromatography or gel filtration. If the disulfide stabilized antibody H chain or disulfide stabilized antibody L chain is insoluble and exists as a granule (i.e., inclusion body), their collection and purification can be performed by lysis of the cells, repeated centrifugation and washing for isolation of the granule, solubilization with guanidine-HCl and subsequent performance of various types of chromatography or gel filtration.
The purified disulfide stabilized antibody H chain and disulfide stabilized antibody L chain are mixed and subjected to a refolding procedure for deriving an active structure (Molecular Immunology, 32, 249 (1995)), thereby forming a disulfide bond. Subsequently, the active disulfide stabilized antibody can be purified by antigen affinity chromatography or ion-exchange chromatography or gel filtration. The activity of the disulfide stabilized antibody can be determined by the method described in 1 (5) or the like.
(3) Method of Use of Disulfide Stabilized Antibody
The disulfide stabilized antibody of the present invention can bind specifically to a human IL-5R xcex1 chain, thereby inhibiting the biological activity of IL-5. Hence, the disulfide stabilized antibody of the present invention is expected to inhibit the function of eosinophils which are controlled in differentiation and growth by IL-5. Accordingly, the disulfide stabilized antibody of the present invention will be useful in the treatment of diseases in which eosinophils are associated with the pathogenesis. The disulfide stabilized antibody of the present invention can be used either alone or in combination with at least one pharmaceutically acceptable adjuvant. For example, the single chain antibody or disulfide stabilized antibody is dissolved in physiological saline or an aqueous solution of glucose, lactose, mannitol or the like to prepare a pharmaceutical composition. Alternatively, the disulfide stabilized antibody is lyophilized by a conventional method and sodium chloride is added to prepare an injection in a powder form. If necessary, the present pharmaceutical composition may contain any additive that is well known in the field of pharmaceutical preparations such as a pharmaceutically acceptable salt and the like.
The present pharmaceutical composition can be administered to mammals, including humans, at a dose of 0.1-20 mg/kg/day of the disulfide stabilized antibody, which may vary depending on the age and condition of the patient and the like. The administration is given once a day (single dose or continuous administration), 1-3 times a week or once every 2-3 weeks by intravenous injection.
5. Method for Detection and Determination of Human Interleukin-5 Receptor xcex1 Chain Using Anti-human IL-5R xcex1 Antibody
(1) Immunocyte Staining Using Anti-human IL-5R xcex1 Antibody
When immunocytes are suspended cells, they are used as such in the following treatment. When immunocytes are adherent cells, they are detached with trypsin in EDTA and then used in the following treatment. The immunocytes are suspended in an immunocyte stain buffer (PBS containing 1% BAS, 0.02% EDTA and 0.05% sodium azide) or the like and dispensed in an amount of 1xc3x97105xe2x88x922xc3x97106 cells. The culture supernatant of the anti-human IL-5R xcex1 monoclonal antibody-producing hybridoma obtained in 1 (4), the culture supernatant of the anti-human IL-5R xcex1 humanized antibody transformant obtained in 2 (9) or the purified antibody obtained in 1 (6) or 2 (9), or the product obtained by labeling the purified antibody with an appropriate labeling substance (e.g., biotin) by a known method (KOUSOKOUTAIHOU (Methods for Enzymes and Antibodies), published by Gakusai Kikaku, 1985) and diluting the labeled antibody with an immunocyte stain buffer or a 10% animal serum-containing immunocyte stain buffer to a concentration of 0.1-50 xcexcg/ml is dispensed in an amount of 20-500 xcexcl and reacted on ice for 30 minutes. When the culture supernatant of the mouse anti-human IL-5R xcex1 monoclonal antibody-producing hybridoma obtained in 1 (4), the anti-human IL-5R xcex1 humanized antibody transformant obtained in 2 (9) or the purified antibody obtained in 1 (6) or 2 (9) has been reacted, the cells are washed with an immunocyte stain buffer after completion of the reaction and an immunocyte stain buffer containing about 0.1-50 xcexcg/ml of an anti-mouse immunoglobulin antibody, anti-rat immunoglobulin antibody or anti-human immunoglobulin antibody which have been labeled with a fluorochrome such as FITC or phycoerythrin is dispensed in an amount of 50-500 xcexcl, followed by reaction on ice for 30 minutes in the dark. When the biotin-labeled monoclonal antibody has been reacted, streptoavidin labeled with a fluorochrome such as FITC or phycoerythrin is dispensed in an amount of 50-500 xcexcl and reaction is performed on ice for 30 minutes in the dark. When the monoclonal antibody labeled with a fluorochrome such as FITC or phycoerythrin has been reacted, an immunocyte stain buffer containing about 0.1-50 xcexcg/ml of the monoclonal antibody is dispensed in an amount of 50-500 xcexcl and reaction is performed on ice for 30 minutes in the dark. In each of these cases, the reaction mixture is washed thoroughly with an immunocyte stain buffer after the reaction and subjected to an analysis with a cell sorter.
(2) Test for Inhibition of Growth of Human IL-5-Dependent Cells Using Anti-human IL-5R xcex1 Antibody
In order to show the biological inhibition activity of the obtained anti-human IL-5R xcex1 antibody, the effect on the growth of human IL-5-dependent cells is examined using human IL-5 dependent cells. Examples of the evaluation method include incorporation of tritium-labeled thymidine into cells, color development methods using cell counting kits and the like. A color development method used in the present invention will now be explained.
CTLL-2 (h5R) cells (1xc3x97104) are suspended in a normal medium (50 xcexcl) and dispensed in a 96-well culture plate. To the plate are added 25 xcexcl of a solution of the purified antibody (0.01-50 xcexcg/ml) obtained in 1 (6) or 2 (9) and a normal medium containing 0.4-40 ng/ml of human IL-5 and the mixture is cultured in a 5% CO2 incubator at 37xc2x0 C. for 24-72 hours. Subsequently, a cell counting kit solution is added at 10 xcexcl/well and the cultivation is continued in a 5% CO2 incubator at 37xc2x0 C. for 4 hours. After completion of the cultivation, the absorbance at 450 nm is determined with a microwell plate reader Emax (Molecular Device) and the CTLL-2 (h5R) cell growth-inhibiting activity of the respective antibody is calculated.
(3) Suppression of Survival of Human Eosinophils by Anti-human IL-5R xcex1 Antibody
Human polymorphonuclear leukocyte fractions which contain eosinophils are prepared from human peripheral blood with a commercially available corpuscle separation medium such as a polymorphprep (Nikomed) or a percoll (Pharmacia). The fractions are suspended in a normal medium and the resulting cells are dispensed in a 96, 48 or 24-well culture plate in an amount of 1xc3x97106xe2x88x921xc3x97107 cells/well, followed by addition of human IL-5 to a final concentration of 0.001-10 ng/ml. The culture supematant of the anti-human IL-5R xcex1 monoclonal antibody-producing hybridoma obtained in 1 (4) or the culture supernatant of the anti-human IL-5R xcex1 humanized antibody transformant obtained in 2 (9) or the purified antibody obtained in 1 (6) or 2 (9) is added and the mixture is cultured in a 5% CO2 incubator at 37xc2x0 C. for 2-5 days. After completion of the cultivation, a cell sample is prepared from each well and stained by May-Grunwald-Giemsa staining method (SENSHOKUHOU NO SUBETE (Techniques for Staining, published by Ishiyaku Shuppan Cor., Ltd., 1988) or the like and the percentage of eosinophils is determined. The absence or presence of the activity of the monoclonal antibody in suppressing the viability enhancement of IL-5-dependent human eosinophils is confirmed by comparing the percentage of eosinophils in the absence of the anti-human IL-5R xcex1 antibody with that in the presence of the anti-human IL-5R xcex1 antibody.
(4) Determination of shIL-5R xcex1 Using Monoclonal Antibody
A plate is coated with 0.1-50 xcexcg/ml of the purified antibody obtained in 1 (6) or 2 (9) as a primary antibody. The coated plate is reacted with 0.1-10,000 ng/ml of the purified shIL-5R xcex1 obtained in 1 (1) or a sample such as human serum. The plate is washed thoroughly and then reacted with a secondary antibody which is an anti-human IL-5R xcex1 antibody recognizing an epitope other than that recognized by the anti-human WL-5R xcex1 antibody which was selected for use as the primary antibody from the purified antibodies obtained in 1 (6) or 2 (9). The secondary antibody was labeled with biotin, an enzyme, a chemiluminescent substance, a radioactive compound or the like prior to the reaction. Subsequently, a reaction is performed in accordance with the label. A calibration curve is constructed on the basis of the reactivity with the purified shIL-5R and the concentration of shIL-5R in the sample is calculated.
(5) Detection of shIL-5R xcex1 by Western Blotting
The purified shML-5R xcex1 obtained in 1 (1) is subjected to SDS polyacrylamide electrophoresis (SDS-PAGE) and then blotted on a polyvinylidene difluoride membrane (hereinafter referred to as xe2x80x9cPVDF membranexe2x80x9d, Millipore). The PVDF membrane is immersed in PBS supplemented with 1-10% bovine serum albumin (BSA) and left to stand at 4xc2x0 C. overnight for blocking, followed by thorough washing with PBS containing 0.05% Tween. The PVDF membrane is immersed in the culture supernatant of the hybridoma obtained in 1 (5) or a solution of the purified antibody obtained in 1 (6) at room temperature for 2 hours and washed thoroughly with PBS containing 0.05% Tween. The PVDF membrane is immersed in a solution of an anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody as a secondary antibody at room temperature for 1 hour and washed thoroughly with PBS containing 0.05% Tween. The secondary antibody was labeled preliminarily with biotin, an enzyme, a chemiluminescent substance, a radioactive compound or the like. After removing the washing solution completely, a reaction is performed in accordance with the label on the secondary antibody and a check is made for the reactivity with a protein which agrees in the molecular weight to the purified shrL-5R xcex1.
(6) Immunoprecipitation of shIL-5R xcex1
An anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody is diluted 10-1000 fold with PBS or other buffer. The dilutions are dispensed in a 96-well ELISA plastic plate at 50-200 xcexcl/well and left to stand at 4xc2x0 C. overnight or at room temperature for at least 2 hours, whereby they are adsorbed on the plate. The plate is washed with PBS. PBS containing 1-10% BSA and the like is dispensed in the plate at 300 xcexcl/well and left to stand at 4xc2x0 C. overnight or at room temperature for at least 30 minutes to achieve blocking. The plate is washed with PBS. The culture supernatant of the hybridoma obtained in 1 (5) or a solution of the purified antibody obtained in 1 (6) (0.01-50 xcexcg/ml) is added at 50-200 xcexcl/well and left to stand at 4xc2x0 C. overnight, thereby adsorbing the antibody on the plate. After the plate is washed, the shIL-5R xcex1 obtained in 1 (1) is diluted with PBS or the like containing 1% BSA to a concentration of 0.1-100 xcexcg/ml and the dilutions are dispensed at 50-200 xcexcl/well, followed by reaction at 4xc2x0 C. overnight. After the plate is washed with PBS or the like containing 0.05% Tween, a 1xc3x97-5xc3x97 sample buffer for SDS-PAGE is dispensed at 50-200 xcexcl/well and shaken at room temperature for at least 30 minutes. After optional dilution with PBS, the solution is added to each lane in an amount of 5-25 xcexcl and subjected to SDS-PAGE, followed by blotting on a PVDF membrane or the like by a conventional method. The PVDF membrane is subjected to western blotting as described in 5 (5), thereby detecting shIL-5R xcex1.